This invention relates to an instrument for determination of the base sequence of nucleic acid, particularly to an instrument for determination of the base sequence of nucleic acid which can increase the number of base sequences to be determined by one run of electrophoresis by detecting nucleic acid fragments with a higher resolving power without impairing the detection sensitivity and thereby detecting longer nucleic acid fragments, as separated from each other.
Heretofore, the base sequence of nucleic acid has been determined according to Maxam-Gilbert method or dideoxy method of preparing nucleic acid fragments of various lengths labeled with a radioisotope, subjecting the fragments to separation by gel electrophoresis according to molecular weight, then visualizing the electrophoretic pattern by autoradiography, and reading the visualized pattern, as disclosed in Saibo Kogaku Vol. 1 (1982), pages 79-87 and 192-202. As a polymer matrix for the gel electrophoresis, there has heretofore been used a polyacrylamide gel in a plate form in which the concentration of buffer is uniform. Such features of the polyacrylamide gel are shown in FIGS. 1a, 1b and 1c.
However, there is recently known a gel in which the concentration of buffer is not uniform but has such a gradient that the concentration of buffer increases, as the buffer is nearer to the end of the migration lane, namely, to the anode, whereby an electrophoretic pattern is reduced at the region nearer the end of the migration lane to separate nucleic acid fragments in a broader range of molecular weight, as disclosed in Proc. Natl. Acad. Sci., U.S.A., Vol. 80, (1983), pp. 3963-3965. Such features of this gel are shown in FIGS. 1d, 1e and 1f.
The above two types of electrophoretic gels have heretofore been used only in a method comprising reading an electrophoretic pattern on the entire gel surface by autoradiography after the completion of electrophoresis (autoradiography method), and no consideration is given to their employment in a method comprising providing a detection part in the migration lane of an electrophoretic apparatus, detecting nucleic acid fragments successively arriving at the detection part by migration, and reading the electrophoretic pattern in real time [real-time direct detection method, see FIGS. 2a, 2b and 2c]. Said electrophoretic gels involve the following problems.
Generally, when nucleic acid fragments to be detected by gel electrophoresis are longer, the distance between the adjacent electrophoretic bands having a difference in length only by the length by which one base will be narrowed. On the other hand, counts of .beta.-ray must be collected to more than a given degree in order to detect the nucleic acid fragments, and hence the slit width cannot be narrowed excessively to reduce the solid angle for effectively detecting the .beta.-ray and lower the detection sensitivity. Employment of the conventional gel having a uniform concentration of buffer shown in FIGS. 1a, 1b and 1c has been disadvantageous in that if the nucleic acid fragments are longer, two or more electrophoretic bands will enter the gel in front of the slit at the same time, so that the individual electrophoretic bands cannot be detected as separated from each other [see FIGS. 3a, 3b and 3c]. The gel having a high concentration of buffer towards the tail end of migration lane, that is, towards the positive electrode shown in FIGS. 1d, 1e and 1f is advantageous in that many base sequences can be determined in the one and same gel when the autoradiography method is employed, but it has been disadvantageous in that when the real-time direct detection method is employed, the distance between the adjacent electrophoretic bands will be rather narrowed, so that it will be more difficult to detect longer nucleic acid fragments, as separated from each other.